Nitric Oxide (NO)Assay Kit (A012)

Instruction manual

First Edition (Revised on April, 2016)

[ INTENDED USE ]

The kit is a nitrate reductase method for the in vitro quantitative measurement of NO in blood serum, gastric juice, urine, cell culture supernates and other biological fluids.

NO has active chemical property, it is converted to nitrate (NO3-) & nitrite (NO2-). As result, it needs amount of blood serum NO3- content plus blood serum NO2- content to express NO level in vivo accurately. Some domestic units use metal cadmium (Cd) reduction method, but this method has tedious operations (protein in blood serum must be removed), low-controllable reactions (cadmium may reduce NO2-further), low determinacy (cadmium can not reduce NO3- to NO2-completely), low accuracy.

Compared with metal cadmium method, nitrate reductase method is a sensitive, convenient, fast, stable, easy-generalized method.

[ REAGENTS AND MATERIALS PROVIDED ]

Reagents

Quantity(50/25T)

Reagents

Quantity(50/25T)

Reagent 1

2×6ml/1×6ml

Reagent 6

1/1

Reagent 2

2×6ml/1×6ml

Reagent 7

1×8mL//1×4ml

Reagent 3

1×12ml//1×6ml

Standard(10mmol/L

1×0.5ml/1×0.5ml

Reagent 4

1×6ml//1×3ml

Attachment

2×40ml/1×40ml

Reagent 5

1/1

Instruction manual

1/1

Note:Attachment is double distilled water.


[ MATERIALS REQUIRED BUT NOT SUPPLIED ]

1.  An spectrophotometer capable of measuring absorbance at 550nm, glass cuvettes of 0.5cm light path

2.  Thermostatic water bath or air bath capable of controlling temperature at 37℃  and 90~100℃ 

3.  Desk centrifuge,

4.  Micropipets and tips

5.  Vortex mixer, glass rod

6.  A source of pure water (preferably double distilled water and double distilled water)


[ STORAGE OF THE KITS ]

1.  Reagent 1: Can be stored at -20℃  or even colder for 3 months. Please place bottle in 37℃  or at room temperature to dissolve completely before use.

2.  Reagent 2: Can be stored at -20℃  or even colder for 3 months. Please place bottle in 37℃  or at room temperature to dissolve completely before use.

3.  Reagent 3: Can be stored at room temperature for 6 months.

4.  Reagent 4: Can be stored at room temperature for 6 months.

5.  Reagent 5: Can be stored away from light.

6.  Reagent 6: Can be stored by cold preservation away from light. If its color becomes dark brown, then please discard it.

7.  Reagent 7: Can be stored at room temperature for 6 months.

8.  10mmol/L standard: Can be stored at -20℃  or even colder for 6 months.

 

[ REAGENT PREPARATION ]

1.  Mixed reagent preparation: Mix Reagent 1 and Reagent 2 at ratio of 1:1, how much you need, how much you prepare. After mixing completely, mixed reagent is available in 24 hours.

2.  Reagent 5: Powder×1 vial, when use, add 20ml*(50T) or 10ml*(25T) 90℃ ~100℃  hot distilled water to dissolve completely, can be stored away from light. Reagent 5 is supersaturated solution, it is better to add 23ml(50T)/11.5ml(25T) hot distilled water (consider thermal expansion), adding, heating by water bath & stirring by glass rod in same time to dissolve completely. There may be crystals in old Reagent 5 (from last experiment), please heat and stir it to dissolve completely.

3.  Reagent 6: Powder×1 vial, when use, add 8ml*(50T) or 4ml*(25T) double distilled water to dissolve it. Can be stored by cold preservation away from light. If its color becomes dark brown, then please discard it.

4.  Chromogenic agent: How much you need, how much you make (if you can exhaust chromogenic agent in 1 month, then you can prepare it in 1 time). Mix Reagent 5, Reagent 6 & Reagent 7 at ratio of 2.5:1:1. Can be stored in 40ml clean square bottle away from light. There are some crystals seed out in cold days, so when you use it again, please place it in 100℃  water bath, dissolve completely by shaking repeatedly.

5.  100μmol/L standard working solution preparation: Take 0.1ml standard, add double distilled water until volume reaches to 10ml (100 times dilution), mix sufficiently, 100 μmol/L standard working solution is prepared. This working solution should be used soon after preparation.


[ SAMPLE PREPARATION ]

1.  Liquid sample (such as blood serum, gastric juice, urine, cell culture supernates and other biological fluids,etc.) NO assay:

Pretreatment: According to Experimental Methodology.

2.  Tissue sample NO assay

Pretreatment: According to Experimental Methodology.


[ ASSAY PROCEDURE ]

1.  Liquid sample NO assay

Operation table:


Blank tube

Standard tube

Sample tube

Double distilled (ml)

0.1

100μmol/L standard working solution (ml)
0.1

Liquid sample (ml)



0.1

Mixed reagent (ml)

0.40.40.4

Mix sufficiently, place in 37℃  water bath for 60 minutes accurately

Reagent 3 (ml)

0.20.20.2

Reagent 4 (ml)

0.10.10.1

Mix sufficiently by vortex for 30 seconds, place quescently for 40 minutes, centrifugate at 3500~4000 rpm for 10 minutes, take supernatant for chromogenic reaction.

Supernatant (ml)

0.50.50.5

Chromogenic agent(ml)

0.60.60.6

Mix sufficiently, place quescently for 10 minutes, transfer in cuvettes of 0.5cm light path, measure OD values of all tubes at 550nm (adjust zero by distilled water)

Note:

a. If you do this assay in winter, then please prewarm all reagents at 37℃  for 5 minutes.

b. If you have not enough supernatant, please extend centrifugation time length or take less supernatant (such as 0.4ml or 0.45ml), but please make sure that all tubes in one batch have sample sample volume. Never add sediment in tubes.

 

2.  Tissue sample NO assay

Operation table:


Blank tube

Standard tube

Sample tube

Double distilled (ml)

0.50.4
100μmol/L standard working solution (ml)
0.1

Homogenate (ml)



0.1

Mixed reagent (ml)

0.40.40.4

Mix sufficiently, place in 37℃  water bath for 60 minutes accurately

Reagent 3 (ml)

0.20.20.2

Reagent 4 (ml)

0.10.10.1

Mix sufficiently by vortex for 30 seconds, place quescently for 40 minutes, centrifugate at 3500~4000 rpm for 10 minutes, take supernatant for chromogenic reaction.

Supernatant (ml)

0.80.80.8

Chromogenic agent(ml)

0.60.60.6

Mix sufficiently, place quescently for 10 minutes, transfer in cuvettes of 0.5cm light path, measure OD values of all tubes at 550nm (adjust zero by distilled water)

 

Note:

a. If you do this assay in winter, then please prewarm all reagents at 37℃  for 5 minutes.

b. If you have not enough supernatant, please extend centrifugation time length or take less supernatant (such as 0.4ml or 0.45ml), but please make sure that all tubes in one batch have sample sample volume. Never add sediment in tubes.


[ TEST PRINCIPLE ]

NO has active chemical property, it is converted to nitrate (NO3 -) & nitrite (NO2-), NO2- can also convert to NO3-. The method use nitrate reductase to reduce NO3- to NO2- selectively, calculate NO content by measuring OD values.


[ REFERENCED SAMPLE VALUE]

1.Optimal sampling volumes:

Bovine blood serum: 300μl

Sheep blood serum: 300μl

Rat & mouse blood serum: 100μl

Rabbit blood serum: 100μl

10% tissue homogenate: 500μl

Cell suspension: 500μl

Cell culture supernates: 100μl

2.Normal NO contents:

表格4

Sample

Referenced sampling volume

Referenced NO content

Rat blood serum

100μl

38.0±23.4μmol/L

Mouse blood serum

100μl

49.96±23.76μmol/L

Rabbit blood serum

100μl

99.16±39.5μmol/L

Dog blood serum

100μl

89.51±32.13μmol/L

[ IMPORTANT NOTE ]

1.  Please avoid free-thawing Reagent 1, Reagent 2 & standard repeatedly. If you want to do experiments in batches (>3), then you can subpack and store Reagent 1, Reagent 2 & standard when you do first experiment, when you do other experiment, take this reagents according to how much you use. Prepared chromogenic agent should be stored away from light.

2.  If you don’t use samples (such as blood serum, blood plasma, tissues, etc.) immediately, then you can store them at -70℃  or even lower temperature. Deepfreezed samples are available in half year.

3.  All reagent preparations and blank tubes need NO2--free double or triple distilled water (This kit includes double distilled water).

4.  After complete reaction and centrifugation, please avoid suck sediment when you take supernatant, or OD values will increase largely, validity of results will be disturbed seriously

5.  Test tube selection:

a. It is suggested to use disposable plastic test tubes (preferably disposable plastic test tubes from our institute).

b. If you want to use glass test tubes, then please soak them in detergent solution for more than half hour, boil for 0.5~1 hour, brush carefully, rinse by tap water for 15~20 times, discard water, rinse by distilled water for 1~2 times, oven dry them.

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